Exposure to ozone causes airway hyperresponsiveness that is mediated via release of acetylcholine from the vagus nerves. Under normal circumstances, acetylcholine release is limited by neuronal M2 muscarinic receptors which in turn inhibits bronchoconstriction. These M2 muscarinic receptors no longer function in guinea-pigs exposed to 0.8-2 ppm ozone for 4 hours. The PI hypothesizes that loss of these receptors increases vagally-mediated bronchoconstriction following ozone exposure and may also contribute to the wheezing, coughing, and shortness of breath experienced by people breathing polluted air. This proposal will address whether environmental concentrations of ozone can alter neuronal M2 receptor function, by determining what minimum concentration of ozone will inhibit neuronal M2 muscarinic receptors in the lung, and whether there is a cumulative effect of exposure to subthreshold concentrations. To identify the mechanisms for ozone-induced loss of neuronal M2 receptor function, the role of inflammatory cells and their products on M2 receptor dysfunction after ozone exposure will be examined. In vivo responses to vagal stimulation will be measured and the effects of depletion of inflammatory cells (via cyclophosphamide), inhibition of eosinophils (using monoclonal antibodies to interleukin-5 and VLA-4), neutralization of positively charged proteins (using heparin and poly-l- glutamic acid) and neutralization of eosinophil major basic protein (using a monoclonal antibody) will be tested. To investigate specific mechanisms and address whether inflammatory cell proteins can alter human muscarinic receptors the Investigators propose to test whether inflammatory cell proteins (such as purified eosinophil proteins, neutrophil homogenates, or interleukin-8 (a cationic, heparin-binding chemokine) can alter receptor number or agonist/antagonist affinity for cloned human muscarinic receptors (m1-m5). In order to assess whether inflammatory cells are actively recruited to the parasympathetic nerves following ozone, immunohistochemistry for the adhesion molecule, VCAM, will be carried out using histological sections from tracheas of ozone- exposed animals. Adherence of inflammatory cells to primary cultures of tracheal parasympathetic nerves will also be measured.